The Non-Ribosomal Peptide Synthetases (NRPSs) are a family of multi-domain enzymes that catalyze the synthesis of peptide antibiotics and peptide siderophores. The NRPSs function as modular assembly lines with amino acids and peptide intermediates bound to the protein and transferred from upstream domains to downstream domains where they are extended and chemically modified. This modular strategy requires large-scale protein rearrangements to allow the nascent peptide to be transferred between domains. The features of the NRPS enzymes that allow this conformational flexibility will be identified through the proposed experiments. A 140-degree domain rotation is hypothesized to allow the multiple states that direct the peptide to different catalytic domains in a coordinated manner. This will be tested through biochemical analyses in which this domain rotation is blocked and multi-domain NRPS enzymes are assayed functionally. Additionally, crystal structures will be determined of conformationally trapped enzymes. These structures will explain how the enzymes adopt the necessary conformations. The long-term objective of this project is the development of new antibiotics. This will be accomplished in two specific ways. First, many NRPS products are peptide antibiotics. There has long been interest in engineering these enzymes to produce novel antibiotics. These experiments have met with limited success because of the uncertainty of the structural basis for catalysis and, more importantly, for the binding specificity of downstream domains. Our experiments will identify crystal structures of highly relevant catalytic states of the enzymes that will explain the active site architecture of catalytic domains that are currently lacking. This information, together with the insights into the conformational dynamics, will enable the engineering of NRPS systems for the production of novel peptides. The second way in which these studies will lead to the development of novel antibiotics relates to the involvement of the NRPSs in siderophore synthesis. These bacterial iron-scavenging compounds serve as important virulence factors in many pathogenic organisms. The inhibition of these pathways is an attractive target for antibiotic design. In a collaborative effort, we will determine the crystal structures of bacterial siderophore-producing NRPS enzymes bound to novel sub micromolar inhibitors. These structures will provide the foundation for optimization of the compounds for improved binding properties. PUBLIC HEALTH RELEVANCE: The NRPS proteins are modular proteins that operate in a fascinating assembly line fashion. A complete understanding of the how these proteins work will enable the production of new antibiotics in two ways. These protein factories can be engineered to make new active compounds. Additionally, these NRPS proteins make bacterial virulence factors needed for infections and we will determine molecular pictures of these enzymes bound to lead inhibitors to allow the design of second generation compounds with improved properties.